title: A cellular replicon-based phenotyping assay to determine susceptibility of hepatitis C virus clinical isolates to NS3/4A protease inhibitors.
abstract: A hepatitis C virus (HCV) replicon-based protease phenotyping assay has been developed that allows determining the susceptibility of a patient's HCV protease sequence to HCV protease inhibitors. In brief, HCV protease sequences amplified from clinical samples are cloned in a transient HCV genotype 1b replicon backbone, containing a luciferase reporter gene. These protease chimeric replicons are replication-competent when electroporated into susceptible Huh7-Lunet cells. Replication can be quantified by measuring the enzymatic activity of the luciferase protein. This assay is reproducible and robust, and has a high overall success rate for determining the phenotypic susceptibility of HCV genotype 1a and 1b patient-derived protease domains to HCV protease inhibitors. In addition, the HCV genotype 1b protease shuttle backbone also supports efficient replication of HCV genotype 4 protease sequences.
authors.affiliation: Janssen Infectious Diseases-Diagnostics BVBA, Johnson & Johnson Corporation, Beerse, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Vijgen
authors.forename: Jannick
authors.initials: J
authors.lastname: Verbeeck
authors.forename: Barbara
authors.initials: B
authors.lastname: Van Kerckhove
authors.forename: Jan Martin
authors.initials: JM
authors.lastname: Berke
authors.forename: Diana
authors.initials: D
authors.lastname: Koletzki
authors.forename: Gregory
authors.initials: G
authors.lastname: Fanning
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D002474:Cell-Free System; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D016174:Hepacivirus / Q000187:drug effects* / Q000302:isolation & purification / Q000502:physiology*; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods*; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000494:pharmacology*; D014158:Transcription, Genetic; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors; D014779:Virus Replication / Q000187:drug effects*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000998:Antiviral Agents; D011480:Protease Inhibitors; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus